The long-term goal of this project is to understand the basic molecular mechanisms and signalling pathways underlying immune tolerance. The specific objectives are to determine whether the transcription factor NFAT plays a role in tolerance induction. The underlying hypothesis to be tested is that tolerance induction involves unbalanced activation of NFAT in the absence of its cooperating transcription factor AP-1 (Fos/Jun). In Aim 1, the requirements for tolerance induction by NFAT will be defined. NFAT1-/- T cells, which we have shown are much less susceptible to the induction of anergy in a cell culture model than are T cells from normal mice, will be reconstituted with wildtype NFAT, an NFAT mutant incapable of interacting with AP-1, and a version of NFAT that is hypersensitive to calcineurin, and the ability of the reconstituted cells to be anergised in a cell culture model will be compared. A selective peptide inhibitor of NFAT (VIVIT) will be tested for whether it interferes with tolerance induction. In Aim 2, the role of NFAT will be tested in physiological systems of tolerance induction, including the high dose antigen model of T cell tolerance and the HL model of B cell tolerance. The selective NFAT inhibitor VIVIT will be transiently expressed in T cells during the first stage of tolerance induction and tested for whether it abrogates tolerance induction in vivo. In Aim 3, NFAT-dependent target genes that are induced in tolerant T cells will be identified using multiple approaches (DNA arrays, subtractive strategies, and genome-wide retroviral insertion). Selected genes will be tested for whether they fit criteria expected for genes involved in tolerance induction. In Aim 4, the role of NFAT in anergy induced by altered peptide ligands will be tested using the approaches and reagents already used in Aim 1. The experiments will determine whether tolerance has a transcriptional basis and whether inhibitors of the NFAT-Fos-Jun interaction can be used in cell culture and in vivo to induce a tolerant state.